We wish to determine the mechanism by which insulin participates in the regulation of protein synthesis in muscle and the character of the defect in protein metabolism that occurs in diabetic animals. We propose to compare the specific activity of amino acids in the intracellular water and in aminoacyl-tRNA (and perhaps also in peptidyl-tRNA) to determine if there is a separate pool of amino acids in muscle that is used for protein synthesis; if there is a separate pool we shall test whether the concentration of amino acids in it is affected by insulin and diabetes. Efforts will be made to identify the partial reactions in the initiation of protein synthesis that diabetic ribosomes cannot carry out in a normal manner; we shall compare the capacity of normal and diabetic ribosomal subunits to form a 40S initiation complex in the reaction catalyzed by EIF-3; and the ability to then add a 60S subunit to form an 80S initiation complex in the presence of EIF-1 or EIF-2 or both. Finally, we will compare the structure of normal and diabetic ribosomes: the proteins of the ribosomes will be labeled in vitro by reductive alkylation with 3H- or 14C-formaldehyde, separated by micro 2-D PGE and the ratio of 3H/14C radioactivity determined so as to allow a calculation of the relative stoichiometry of individual proteins in normal and diabetic ribosomes. In separate experiments we propose to use affinity analogues to identify the proteins contained in the aminoacyl-tRNA and mRNA binding sites on the ribosomes and to ascertain if those sites are altered by diabetes or insulin.